Green synthesis of spermine coated iron nanoparticles and its effect on biochemical properties of Rosmarinus officinalis

In this study, aqueous spinach extract was used for the green synthesis of iron nanoparticles. The surface of iron oxide nanoparticles was coated with spermine. The physicochemical properties of nanoparticles were investigated using UV-Vis, TGA, FTIR, VSM, TEM, and DLS. The results showed that the nanoparticles had a spherical structure. The surface charge of the Fe3O4-NPs increased from −3.2 to 18.42 (mV) after Fe3O4 coating by spermine. In order to investigate the effect of nanoparticles on physicochemical properties of rosemary under drought stress conditions, an experiment was carried out in a completely randomized design. The results showed that the amount of antioxidant enzymes and secondary metabolites increased significantly under drought stress. Moreover, the use of spermine-coated iron nanoparticles can be useful in increasing resistance to drought stress in plants by increasing the activity of some antioxidant enzymes and secondary metabolites. The biocompatibility of Nanoparticles in cell suspension was investigated. the ability of Fe3O4-SM NPs to interact with DNA and protect it against DNaseI and ultrasonic waves using agarose gel electrophoresis was studied. The ability of Fe3O4-SM to neutralize the negative charge of DNA and protect it against DNaseΙ and ultrasonic waves was confirmed using an agarose gel electrophoresis assay.

Iron oxide nanoparticle synthesis. Briefly, 3.33 g of iron (III) chloride hexahydrate (FeCl 3 . 6H 2 O) and 1.59 g of iron (II) chloride tetrahydrate (FeCl 2 . 4H 2 O) were dissolved with 100 ml of deionized. The solution was mixed under nitrogen gas at 80 °C for 10 min. Subsequently, 15 ml of hydroalcoholic spinach extract was added dropwise into the mixture under continuous stirring for 10 min. After five minutes, 60 ml of NaOH 1M was added to the solution. The reaction continued until the color of the solution changed from yellow to dark brown. Then the nanoparticles were collected using a magnet and then washed with deionized water to remove impurities. Fe 3 O 4 @SiO 2 -SS-SM nanoparticles synthesis. The Fe 3 O 4 @SiO 2 -SS-SM nanoparticle synthesis was performed using the method reported by Zhang et al. (2016) 21 . To synthesize the Fe 3 O 4 @SiO 2 -SH, 200 mg of Fe 3 O 4 nanoparticles were dispersed in ethanol by sonication for 15 minutes, followed by sequential addition of 1 mL NH 3 .H 2 O, 1 mL MPTMS and 20 mL TEOS to the ultrasound water bath. Finally, the nanoparticles were separated using a magnet, washed five times with ethanol and water, and vacuum-dried overnight. The resulting Fe 3 O 4 @SiO 2 -SH was then conjugated with COOH with 200 mg of sulfhydrylated Fe 3 O 4 nanoparticles dispersed in 12 mL methanol and were mixed with equal amounts of 2-carboxyethyl 2-pyridyl disulfide. The reaction was maintained for 36 hours, and the product was then washed and dried, as discussed earlier.
To synthesize the Fe 3 O 4 @SiO 2 -SS-SM nanoparticles, 200 mg of Fe 3 O 4 @SiO 2 -SS-COOH nanoparticle was dispersed in 16 mL PBS buffer (pH = 7.4). Then, NHS (10 mg) and EDC (20 mg) were added at room temperature for 1 hour to activate the carboxyl terminal for Fe 3 O 4 @SiO 2 -SS-COOH. After that, the mixed solution was added to 4 ml of PBS buffer that contained 200 mg of spermine and was stirred at 25 °C for two days. The final product of nanoparticles was washed several times with de-ionized water and ethanol and dried in a freeze drier. Figure 1, Shows a schematic of Fe 3 O 4 -SM nanoparticles synthesis.
Rosmarinus officinalis genomic DNA extraction. Genomic DNA extraction from Rosmarinus officinalis was performed as described in the previous report 22 . Retardation assay. To interact DNA with Fe 3 O 4 -SM nanoparticles, a fixed amount of Rosmarinus officinalis DNA (5 µg) was combined with different concentrations of Fe 3 O 4 -SM nanoparticles (0, 0.25, 0.5, 1, 3, 4, and 5 mg) and then gently vortexed for 30 sec. The Fe 3 O 4 -SM/DNA complex was incubated at room temperature for 30 minutes. Finally, the interaction ability of nanoparticles with DNA was investigated by agarose gel electrophoresis. DNA was visualized using electrophoresis in 0.8% agarose gel at 80 V for 2 h, then stained with ethidium bromide and observed under UV light.    Table 1).
Estimation of the terpenes content of Rosmarinus officinalis leaves using high-performance liquid chromatography (HPLC). High-performance liquid chromatography was used to investigate the effect of Fe 3 O 4 and Fe 3 O 4 -SM nanoparticles on the Terpenes content of Rosmarinus officinalis. Briefly, one g of powder from leaves of Rosmarinus officinalis was extracted several times with 20 mL methanol. Next, the extracts were filtered and concentrated using a vacuum incubator. 20 µl of each extract was applied on HPLC (KNAUER-Germany). The stationary phase was an L10 column (Nitrile), and the flow rate was 0.5 mL/min 35 . UV detection (K2500) was used at λ = 275 nm for α-pinene and 315 nm for camphene ،α-terpinene and 1,8-cineol. 1 mg/ml of pure α-pinene, camphene, α-terpinene, and 1,8-cineol were used separately as HPLC standards. The elution  www.nature.com/scientificreports/ solvent was composed of water containing 0.2 sulfuric acid (Solvent A) and methanol containing 0.2 sulfuric acid (Solvent B). Each Terpenes compound was quantified using a calibration curve prepared with each standard (Sigma-Aldrich, USA) and a co-chromatogram of the standards and samples. The experiments were repeated thrice, and each sample was assayed in triplicate 36,37 .
Statistical analysis of the data. Each treatment was conducted with three replicates and comparisons concerning treatment tools were made by recruiting the least significant difference (LSD) at the 0.05 and 0.01 probability levels by SAS 9.4 software and Excel application software. The standard deviation of means ± was then calculated from the average of each treatment. The UV/Vis curve of Fe 3 O 4 nanoparticles synthesized with spinach extract is reported in Figure 2B. As shown in Figure 2B, a strong peak in the range of 350-400 nm confirms that Fe 3 O 4 is synthesized and stable. In addition, the absorption peak confirms the synthesis of Fe 3 O 4 in nanometric dimensions. The UV/Vis results indicate no visible peak for spermine, and this result was consistent with the results of previous reports 38 . Meanwhile, the UV/Vis spectra showed that the peak of Fe 3 O 4 @SiO at 350-400 nm sharply decreased because of the spermine binding on the surface of the Fe 3 O 4 @SiO nanoparticle ( Figure 2B).

Fourier-transform infrared spectroscopy (FT-IR).
FT-IR technique was used to confirm the synthesis of nanoparticles and the presence of possible functional groups on the iron oxide nanoparticles synthesized with spinach extract ( Figure 2C). The peaks at 575 cm −1 indicate the tensile vibration of the octahedral Fe-O structure, and the peak at 2931 cm −1 is related to the C-H bond tensile vibration. The peak at 575 cm −1 confirms the presence of the Fe 3 O 4 compound and proves that no other compound of iron e.g., goethite or hematite, has been synthesized besides this one. The FT-IR spectra for the Fe 3 O 4 @SiO 2 -SH and Fe 3 O 4 @SiO 2 -SS-SM are illustrated in Figure 2D. As can be seen, the Fe 3 O 4 @SiO 2 -SH presented peak at 11050 cm −1 is related to the Si-O bond of Fe 3 O 4 @SiO 2 -SH nanoparticles.
The peaks at 1645-1755 cm −1 in the FT-IR spectra for the Fe 3 O 4 @SiO 2 -SS-SM confirmed the conjugation of SM to the Fe 3 O 4 @SiO 2 -SS-COOH. Once the SM is conjugated with the Fe 3 O 4 @SiO 2 -SS-COOH, the SM peaks from 1645 to 1755 cm −1 , which can be attributed to the amide and amide amino groups will indicate the attachment of SM to the surface of Fe 3 O 4 @SiO 2 -SS-COOH nanoparticles ( Figure 2E The broadening of the XRD peaks indicates the small size of the synthetic magnetite. The size of the synthesized nanomagnetite crystals was calculated using the characteristic peaks through the Debye-Scherer equation. www.nature.com/scientificreports/ The average particle size was calculated to be about 18 nm at the main peak locations. This pattern also confirmed the cubic structure of synthetic nanoparticles.

The images of transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The TEM images of the Fe 3 O 4 and Fe 3 O 4 -SM nanoparticles showed that the obtained nanoparticles
were about 10-40 nm in size, which was congruent with the results of DLS. The nanoparticles also had a spherical structure consistent with the results of previous research. After coating the iron oxide nanoparticles with spermidine, the particle size increased slightly, and in addition to the spherical structure, oval structures were observed ( Figure 3). The morphology of nanoparticles has a significant effect on their properties. According to past research, spherical nanoparticles have a higher transfer rate to cells than other nanoparticles' morphologies 43,44 . Also, SEM images of the synthesized Examining the ability of Fe 3 O 4 -SM nanoparticles to interact and protect DNA. The present study results showed that the amine groups in spermine can interact electrostatically with the negative charge of the phosphate group in DNA due to their cationic charge. As shown in Figure 5B, there was no significant difference in the DNA banding pattern between the naked DNA and the DNA/Fe 3 O 4 -SM nanoparticles were prepared at the mass ratios lower than that of 1000 of the nanoparticles to 1 of the DNA (% w/w). However, no DNA bands were observed in the agarose gel in the mass ratio of 3000 Fe 3 O 4 -SM nanoparticles to 5 of DNA (% w/w) and above. These results show that by neutralizing the negative charge of DNA by Fe 3 O 4 -SM nanoparticles, the migration toward the positive polar of agarose gel is completely stopped and remains in the well ( Figure 5B). As shown in Figure 5C;  www.nature.com/scientificreports/ and ultrasound ( Figure 5D). DNA-coated nanoparticles appear to protect DNA from ultrasonic waves like a shield. Also, the binding of nanoparticles to DNA causes it to be coated, thus preventing the binding of restriction enzymes to DNA 23 .  Figure 6).

The effect of drought stress and Fe 3 O 4 -SM application on biochemical parameters of Rosmarinus officinalis. Based on variance analysis, the interaction effects of nanoparticles (Fe 3 O 4 and Fe 3 O 4 -SM)
and drought stress were significant in the biochemical parameters of Rosmarinus officinalis (Table 2). Results showed that the soluble sugar and proline content of Rosmarinus officinalis increased significantly under drought stress. The results also showed that these amounts even increased after applying iron oxide and spermine-iron oxide nanoparticles to Rosmarinus officinalis. In fact, among the treatments, the application of 50 mg/L of Fe 3 O 4 -SM in drought stress (FS 50 I 1 ) led to an increase in proline and soluble sugars, compared to the control treatment (Table 3). Also, similar trends were observed for anthocyanin content. As shown in Table 3, the highest anthocyanin content was observed with treatment with 100 mg/L of Fe 3 O 4 -SM under drought stress conditions (FS 100 I 1 ) (80.4±7.5 µM/g FW). In other words, anthocyanins are part of phenolic compounds and form a large group of secondary metabolites; therefore, while having antioxidant properties, they act as free radical receptors and protect plants against oxidative stress 48 . Considering that the highest amount of anthocyanin was obtained under drought-stress conditions, the explanation for this rise can be linked to the photoprotective action of anthocyanin through the elimination of reactive oxygen species during oxidative stress. Plants are a rich source of phenolic compounds, which are the most important natural and secondary antioxidants. Therefore, among the secondary metabolites, we can mention the phenolic-flavonoid compound, which increases under www.nature.com/scientificreports/ conditions of oxidative stress. As a result, the highest amounts of total phenol (1.9±0.14 mg/g FW) and flavonoid content (41.1±4.8 mg/g FW) were observed in the applications of 100 mg/L (FS 100 I 1 ) and 50 mg/L of Fe 3 O 4 -SM in drought stress conditions (FS 50 I 1 ), respectively. Plants appear to boost the production of secondary metabolites such as phenolic compounds and anthocyanin in response to drought stress to cope with the impacts of reactive oxygen species and adapt to new conditions. The researchers reported that drought stress leads to an increase in secondary metabolites, including anthocyanins, phenols, and flavonoids 49 , which is consistent with the present study. Furthermore, the effects of drought stress and spermine-iron oxide nanoparticle applications on Rosmarinus officinalis were investigated by measuring 1,1-diphenyl-2-picrylhydrazyl (DPPH) in a methanolic extract of Rosmarinus officinalis. The IC50 value is the amount of extract required to scavenge 50% of the DPPH radical. therefore, the decrease in the IC50 value of the extract may reflect its potent antioxidant properties. The lowest concentration required for scavenging 50% of the DPPH radical was observed in FS 100 I 0 (110.51±9.5 µg/g FW). Therefore, it can be concluded that the application of Fe 3 O 4 -NPs by spermine improves plant stability against free radicals (Table 3). On the other hand, the analysis of variance showed that the effects of nanoparticles and drought stress significantly affected protein content ( Table 2). As can be seen in Table 3, the highest content of protein was (0.43±0.03 mg/g FW) that was obtained from 100 mg/L Fe 3 O 4 -SM (FS 100 I 0 ) in complete irrigation conditions, and the lowest content of protein was in no application of nanoparticles in drought stress conditions (0.28±0.01 mg/g FW).
The absence of nanoparticles and the drought stress condition reduced the content of protein by approximately 53.57% when compared to the application of 100 mg/L Fe 3 O 4 -SM in complete irrigation conditions. The decrease in protein content under drought stress appears to be caused by the reaction of protein with free radicals, which results in amino acid changes, a decrease in protein synthesis, an accumulation of free amino acids, including proline, and an increase in the activity of protein degrading enzymes. Furthermore, when 100 mg/L Fe 3 O 4 -SM was used, the soluble proteins increased significantly. It seems that the increase of soluble proteins in the application of Fe 3 O 4 -NP coating with spermine is due to the synthesis of new proteins, the increase in the level of proteins related to stress tolerance, such as proline, or the role of this nanoparticle in dealing with oxidative stress. Also, The effect of Fe 3 O 4 -NP and spermine in preventing the structural and functional destruction of the  (Table 4). As shown in Figure 7A, the application of Fe 3 O 4 -NPs and Fe 3 O 4 coating by spermine had significant effect on the hydrogen peroxide content (H 2 O 2 ) of Rosmarinus officinalis. Drought stress significantly increased the hydrogen peroxide content in Rosmarinus officinalis. The increase in levels of H 2 O 2 content under drought stress has also been reported in previous studies 51, 52 . This increase depends on the severity of the drought stress   Figure 7D). It has been reported that the use of Fe 3 O 4 -NPs and spermine increases the activity of some important enzymes participating in the oxidative defense system, such as catalase and peroxidase, and decreases H 2 O 2 in plants that are under drought stress 53 . Also, it has been reported that H 2 O 2 , as a regulatory factor, plays an important role in the activation of genes encoding proteins that are involved in the defense against oxidative stress 54 . Also, it has been reported that under conditions of drought stress, the content of hydrogen peroxide increases, and antioxidant enzymes are the most important compounds in deactivating free radicals 55 . The antioxidant role of polyamines has already been established. Polyamines decrease the levels of reactive oxygen species (ROS) in cells by increasing antioxidant enzyme activity 56 . It has been reported that the use of polyamines, including spermine, leads to the reduction of H 2 O 2 in conditions of environmental stress 57 .
Our results showed that drought stress significantly increased the activity of antioxidant enzymes. In addition, it was found that coating Fe 3 O 4 nanoparticles with spermine significantly increased the activity of antioxidant enzymes compared to Fe 3 O 4 nanoparticles. So, among the treatments, the highest activities of ascorbate peroxidase, polyphenol oxidase, and catalase enzymes in Rosmarinus officinalis were obtained under drought stress conditions ( Figure 7B-D). Indeed, environmental stress affects the plant's activity. The current study demonstrates that an increase in antioxidant enzyme activity is one of the important mechanisms that occurs when the plant experiences environmental stresses, such as drought stress, to increase the plant's tolerance to these conditions. An increase in the activity of antioxidant enzymes such as catalase ascorbate and peroxidase can be considered a cellular defense mechanism against oxidative damage under stress conditions 58 . Also, the results showed that the use of Fe 3 O 4 coated with spermine led to an increase in the activity of these antioxidant enzymes compared to no application of nanoparticles. In other words, the application of 100 mg/L of Fe 3 O 4 coated with spermine led to an increase in the ascorbate peroxidase (466 μmol min −1 mg −1 ), polyphenol oxidase (639 μmol min -1 mg −1 ), and catalase (16.03 μmol min −1 mg −1 ) enzymes in Rosmarinus officinalis under drought stress conditions ( Figure 7B-D). In other words, the application of 100 mg/L of Fe 3 O 4 coated with spermine led to an increase of 36.35%, 70.35%, and 93.36% respectively in polyphenol oxidase, catalase, and ascorbate peroxidase compared to the control treatment. One of the reasons for the increased activity of antioxidant enzymes in drought-stress conditions can be due to the use of Fe 3 O 4 coated with spermine compared to its non-use. Because Fe 3 O 4 -SM protects the cell membrane against lipid peroxidation, it makes the plant tolerate stress conditions and increases the activity of antioxidant enzymes such as catalase and ascorbate peroxidase. Table 3. Means comparison of biochemical traits as affected by nanoparticles and drought stress F 0 , F 50, and F 100 : Iron oxide levels (0, 50 and 100 mg/L respectively), FS 0 , FS 50 and FS 100 : spermine-iron oxide levels (0, 50 and 100 mg/L respectively), I 0 and I 1 : Irrigation levels (complete irrigation and drought stress respectively). www.nature.com/scientificreports/ Also, as shown in the mean comparison results the highest content of α-pinene (14.25 mg/g DW) and α-terpinene (49.29 mg/g DW) were observed in the application of 100 mg/L of Fe 3 O 4 coated with spermine under drought stress (FS 100 I 1 ) that showed a non-significant difference with FS 50 I 1 treatment ( Figure 7E,F). It has been reported that drought stress can increase the concentration of secondary metabolites and increase the expression of genes involved in the synthesis of these metabolites in medicinal plants 59 . Also, the results showed that under drought stress conditions, it leads to a significant increase of l,8-cineol, and camphene. On the other hand, the use of treatments FS 50 and FS 100 also had a positive effect on the increase of l,8-cineol, and camphene compared to the control treatment ( Figure 7G,H). Generally, the results showed that drought stress increased the secondary metabolite content in Rosmarinus officinalis. Phenolic compound levels in Rosmarinus officinalis www.nature.com/scientificreports/ also increased after treatment with spermine-iron oxide nanoparticles. The results of our study are in agreement with previous studies. Previous reports have shown that under various stresses, plant energy is used to produce secondary metabolites to help to prevent cellular damage by free radicals 60 .

Conclusion
Plants try to keep themselves in ideal conditions under stress. Therefore, many plant metabolites change quantitatively and qualitatively in this respect. What was observed in this study also confirms this fact. Different plant compounds show different responses to various stresses. The results of the present study showed that the effect of environmental stress on the increase of secondary metabolites in plants, and the expression of relevant genes in different tissues may be affected differently. The results showed that the effect of drought stress in increasing secondary metabolites such as l,8-cineol, camphene, and α-terpinene in Rosmarinus officinalis is intensified by the application of iron oxide nanoparticles and spermine-coated iron oxide nanoparticles. The results also showed that the simultaneous effects of iron oxide nanoparticles and spermine-coated iron oxide nanoparticles increased the activity of some antioxidant enzymes and secondary metabolites in Rosmarinus officinalis.
Polyamine compounds, such as spermine, can enhance the pharmacological potential of this plant by further stimulating the biosynthesis of secondary metabolites, such as glycosides, flavonoids, phenols, etc. In general, it seems that the application of spermine-coated iron oxide nanoparticles can be a suitable method to reduce the effects of drought stress.

Data availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.